WO1998046210A1 - Procede et composition pour l'administration ciblee de composes - Google Patents

Procede et composition pour l'administration ciblee de composes Download PDF

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Publication number
WO1998046210A1
WO1998046210A1 PCT/US1998/007560 US9807560W WO9846210A1 WO 1998046210 A1 WO1998046210 A1 WO 1998046210A1 US 9807560 W US9807560 W US 9807560W WO 9846210 A1 WO9846210 A1 WO 9846210A1
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Prior art keywords
microspheres
solution
compounds
composition
present
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PCT/US1998/007560
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English (en)
Inventor
Mohammad W. Katoot
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Katoot Mohammad W
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Application filed by Katoot Mohammad W filed Critical Katoot Mohammad W
Priority to AU71193/98A priority Critical patent/AU7119398A/en
Publication of WO1998046210A1 publication Critical patent/WO1998046210A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2422—Alpha-amylase (3.2.1.1.) from plant source
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0241—Containing particulates characterized by their shape and/or structure
    • A61K8/025—Explicitly spheroidal or spherical shape
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/56—Compounds, absorbed onto or entrapped into a solid carrier, e.g. encapsulated perfumes, inclusion compounds, sustained release forms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/60—Particulates further characterized by their structure or composition
    • A61K2800/65—Characterized by the composition of the particulate/core
    • A61K2800/652—The particulate/core comprising organic material

Definitions

  • the present invention relates to novel microspheres and novel methods employing microwaves for making these microspheres. More particularly, the microspheres may be used to deliver desired cosmetic and therapeutic compounds to desired sites of action in patients or in vitro.
  • Insoluble polypeptide or protein microspheres containing therapeutic compounds which enable the controlled release thereof in biological systems have generated significant interest.
  • Albumin has been a preferred protein or polypeptide for the preparation of such microspheres since it is a naturally occurring product in human serum.
  • Previous methods for preparing protein microspheres result in the formation of relatively hydrophobic spheres which usually require further chemical treatment, and, due to their composition, it is extremely difficult to "load" large quantities of water soluble, biologically active agents or other materials within the microspheres after their synthesis.
  • Recent methods of making relatively hydrophilic microspheres permit loading of water soluble, biologically active agents or other materials within the microspheres after synthesis (Goldberg et al., U.S. Patent No.
  • microspheres Many methods of making microspheres involve use of cross-linker agents and other solvents and surfactants which are potentially injurious to living organisms. What is needed is a novel method of making microspheres which minimizes contamination of the microspheres with these potentially dangerous chemicals.
  • the present invention addresses problems described above by providing a novel microsphere and a novel method for making microspheres.
  • the present invention addresses problems described above by providing a method for making versatile hydrophilic polypeptide microspheres. These microspheres may be loaded to high concentrations with cosmetic or therapeutic compounds.
  • the microspheres of the present invention show higher potency and diminished side effects for the compounds delivered in these microspheres.
  • the hydrophilic protein microspheres of the present invention can be complexed physically or covalently with therapeutic agents. Unlike liposome and other drug carriers used today, microspheres of the present invention do not require special drug conjugates, are considerably less expensive, and are readily prepared from any desired peptide or protein.
  • the microspheres of the present invention may also be synthesized with specific compounds such as molecules inserted on or in their surface to facilitate recognition by a target site. For example, microspheres synthesized with specific molecules on their surface may be selectively recognized by certain cells with receptors or acceptors on their surface, and subsequently internalized.
  • the microspheres of the present invention may also be designed with molecules on their surface for recognition and binding by subcellular components.
  • Intracellular delivery of compounds such as translation inhibitors to the endoplasmic reticulum or delivery of mitotic inhibitors to the nucleus may be achieved with the microspheres of the present invention.
  • the present invention provides novel microspheres and methods for making these microspheres.
  • the microspheres of the present invention may be loaded to high concentrations with therapeutic and/or cosmetic compounds.
  • the present invention provides a novel method of making microspheres that employs microwaves. This microwave-based method decreases potential contamination of microspheres with cross-linking agents, surfactants and other potentially injurious chemicals. These microspheres may be loaded to high concentrations with therapeutic and/or cosmetic compounds.
  • the microspheres of the present invention have been used to subcutaneously and intramuscularly administer effective amounts of peptide hormones, such as insulin, in vivo. These insulin-containing microspheres rapidly affect blood glucose levels and are more dose-effective than administration of free insulin in the absence of microspheres.
  • the microspheres of the present invention possess characteristics that provide controlled release of the compound.
  • Such controlled release includes specific parameters for the amount of compound released at any time and also for the duration of release.
  • Controlled release may also include the release of compounds initiated by some signaling event. Signaling events may include, but are not limited to, a change in the tonicity of the fluid surrounding the microsphere, a change in the ionic environment of the microsphere, and the binding of a molecule on the surface of the microsphere to another molecule such as a cell surface molecule, for example, a receptor.
  • the present invention provides novel microsphere compositions and novel methods of making these microspheres.
  • the microspheres of the present invention may be delivered in high concentrations to selected targets.
  • a feature of the present invention is that microspheres may be designed for stability at selected pH levels, temperature and/or osmotic conditions, and for dissolution at a different specific pH levels, temperature and/or osmotic conditions.
  • a further object of the present invention is to provide microspheres to deliver compounds for therapeutic purposes.
  • Another object of the present invention to provide microspheres to deliver compounds for cosmetic purposes.
  • Another object of the present invention to provide microspheres to deliver compounds for agricultural purposes.
  • Another object of the present invention is to provide microspheres to deliver compounds for treating abnormalities in hormone secretion.
  • Yet another object of the present invention is to provide microspheres to deliver compounds for treating diabetes.
  • Another object of the present invention is to provide microspheres to deliver compounds for treating abnormalities of the immune system.
  • Still another object of the present invention is to provide microspheres to deliver compounds to intracellular sites.
  • Another object of the present invention is to provide microspheres to deliver compounds to nuclei of cells.
  • Another object of the present invention is to provide microspheres to deliver genetic material for gene therapy.
  • Figure 1 Effect of subcutaneous injection of albumin microspheres (diamond), albumin microspheres containing bovine insulin (closed circle - 7.5 mg), or saline
  • FIG. 1 Effect of intramuscular injection of albumin microspheres (diamond), albumin microspheres containing bovine insulin (closed circle - 7.5 mg), or saline
  • the present invention provides a novel method for making microspheres.
  • the method of the present invention substantially improves previous methods of delivery of compounds from microspheres by minimizing the hydrophobic character of microspheres, by increasing the amount of compound which may be loaded into the microsphere, and by decreasing chemical treatment of microspheres.
  • the present invention provides methods for making microspheres that may be loaded with various concentrations of a desired compound or of several desired compounds.
  • the present invention provides microspheres that may be designed to release the compound at a desired rate and for a desired duration.
  • the present invention provides novel methods for making microspheres using microwaves. These microspheres may be loaded with high concentrations of a desired compound and are exposed to reduced levels of potentially injurious chemicals, such as crosslinking agents and surfactants.
  • microsphere is used herein to mean a sphere with a diameter in the range of 10 nm to 100 ⁇ m.
  • the microspheres may contain one or more compounds.
  • Microspheres generally include protein, polypeptides, lipids, phospholipids, or peptides (native or synthetic), or mixtures thereof, optionally complexed with another compound or mixtures of different compounds.
  • compound is used herein to mean any therapeutic compound, cosmetic compound, or agricultural compound which may be placed within or on the surface of a microsphere. It is to be understood that the terms compound and composition are used interchangeably within this application. Compounds include therapeutic and/or cosmetic compounds.
  • the term compound includes, but is not limited to the following: bioactive molecules, cells, drugs, prodrugs, vaccines, anticancer drugs, immunogens, recombinant molecules (including recombinant growth hormone, growth factors, stem cell stimulating factors, especially white cell and red cell stimulating factors), prohormones, hormones, plant hormones, proteins, glycoproteins (including but not limited to growth hormone, luteinizing hormone, thyroid stimulating hormone, follicle stimulating hormone and all other pituitary hormones), steroids (including, but not limited to, testosterone, estrogen, progesterone, giucocorticoids and analogs thereof), pheromones, insecticides, fertilizers, herbicides, contraceptives, neurotransmitters, metabolites, receptors, receptor ligands, receptor agonists and antagonists, peptides, neuropeptides, peptide conjugates, peptide analogs, lipids, fatty acids, carbohydrates, nucleic acids, DNA, RNA, antisense molecules, plasmids, artificial
  • compound includes naturally occurring compounds as well as synthetic variants of these compounds.
  • compound also includes agonists and antagonists of molecules which exert some activity at a desired site.
  • compound also includes fragments of cells including fragments of the cellular membranes, tumor marker molecules, and other compounds that might be used for various purposes, including but not limited to use for generation of antibodies against these compounds. Such compounds might be on the surface of or embedded within the microsphere.
  • the term compound also includes, but is not limited to, lecithins, emollients, humectants, moisturizers, skin penetrants, polymers, polyoxypropylene-polyoxyethylene polymers, sunscreens, ultraviolet radiation blockers, para aminobutyric acid, oil, emulsions, microemulsions, glycerin, fatty acids, esters of fatty acids, ceramides, lipids, pH indicators, collagen, collagen fragments, germicides, stearic acid, glycerin, cholesterol, isopropyl myristate, isopropyl palmitate, triethanolamine, paraben, methyl paraben, polysorbate, lanolin, squalene, propylene glycol, and dimethicone.
  • the term "site of action” is used herein to mean the site at which the compound is active.
  • targeted delivery is employed to mean the delivery of microspheres or their compounds to a desired location. Delivery may be targeted to a site of action of a compound. Delivery also may be targeted to a cell which contains an intracellular target site which may be the site of action of the compound.
  • the microspheres of the present invention may be designed with desired molecules on their surface for recognition by the target site, such as a cell, an organelle, a synapse or a receptor. Targeted delivery also encompasses delivery of a microsphere to a location which then provides access to a site of action.
  • microspheres delivered into a vessel distributing tributaries to a specific vascular bed may be distributed to cells receiving vascular supply from the vascular bed.
  • Delivery includes, but is not limited to, topical parenteral and alimentary delivery.
  • the term "functional group" is used herein to mean any chemical group that may be used for covalent or ionic binding or attachment of compounds. Such functional groups include, but are not limited to the following; amino, amide, imino, hydroxyl, aldehyde, carboxyl, sulfhydryl, and thiol groups.
  • cross-linker is used herein to mean any molecule that effectively links a functional group to a compound or cross-links the matrix material. Any cross- linker such as aldehydes, formaldehyde, glutaraldehyde, carboimide, carbodiimide or others known to those skilled in the art, or combinations thereof, may be used in the practice of the present invention.
  • the targeted delivery of the microspheres of the present invention may involve topical application, subcutaneous injection, or intradermal injection.
  • microspheres may be delivered in vehicles including but not limited to creams, emulsions, mousses, sprays, aerosol sprays, lotions, gels, rinses, shampoos, transdermal patches and ointments. These delivery vehicles may optionally be combined with various fragrances, preservatives, bacteriostats, coloring agents and other therapeutic and cosmetic compounds.
  • load and " situ loading” are used herein to mean the process of placing compounds within the microspheres of the present invention, on the surface of the microspheres, or in the surface of microspheres during their synthesis.
  • post-load is used herein to mean the placement of compounds within the microspheres, on the surface of the microspheres, or in the surface of microspheres after their synthesis.
  • patient is employed to mean any human, animal, insect, plant or other living organism.
  • biosensor is used herein to mean a mechanism which will respond by chemical or physical means to cause a biological response.
  • the present invention provides a unique and versatile protein microsphere technology.
  • the microsphere synthesis is rapid, versatile, and provides microspheres with diameters as small as 10 nm with desirable sizes ranging from approximately 0.01 ⁇ m to 100 ⁇ m, preferably approximately 0.05 ⁇ m to 20 ⁇ m, and most preferably approximately 0.1 ⁇ m to 10 ⁇ m.
  • the microspheres of the present invention are optionally hydrophilic and can be complexed or chemically modified to carry a variety of compounds such as cosmetic compounds, therapeutic compounds, agricultural compounds or combinations thereof, and to provide a unique targeted and controlled compound release delivery system.
  • the present invention has the ability to achieve prolonged and effective concentrations of compounds in the skin, blood stream, gastrointestinal system, reproductive system, nervous system, lymphatic system, peritoneal fluid, cerebrospinal fluid, and in organs, tissues, cells, organelles or in various body compartments, to localize high concentrations of compounds such as drugs in specific areas of the body, to enhance vaccine efficacy, to reduce drug toxicity and other harmful side effects, and to enhance drug stability.
  • the microspheres of the present invention also have several applications in the veterinary and agricultural fields. For example, these microspheres may be used to deliver fertilizers, herbicides, pesticides, insect and animal pheromones, and insect contraceptives.
  • the present invention provides a method for producing hydrophilic polypeptide microspheres which will accept high "loadings" (up to approximately 60% by weight) of compounds. These compounds may be added during or after the microsphere synthesis.
  • the present invention provides a method for producing microspheres that does not require the utilization of surfactants to enable the preparation of highly concentrated dispersions.
  • attachment of compounds including, but not limited to, proteins, growth factors, growth inhibitors, receptors, binding proteins, carbohydrates, glycoproteins, cell surface recognition molecules, lectins, enzymes, antibodies, immunostimulants, immune system recognition molecules, and other compounds to the microspheres of the present invention can readily be accomplished to alter their properties and to design microspheres for specific applications.
  • the microspheres of the present invention may be made that have either tissue-specific or tissue non-specific binding characteristics.
  • Microspheres of the present invention may also be designed for targeting delivery to subcellular compartments or organelles.
  • proteins and polypeptides can be used in making the microspheres and forming the matrix of the microspheres.
  • proteins include, but are not limited to, serum albumin, casein, whey, poly-L-lysine, poly-L-arginine, poly-L-histidine, polyglutamic acid, and any water soluble protein with functional amine groups.
  • proteins include, but are not limited to, enzymes, immunoglobulins, peptides, and glycoproteins.
  • the method of the present invention also gives rise to high concentrations of free reactive functional groups, which render the microspheres capable of being much more highly “loaded” with additional compounds such as cosmetically or therapeutically active compounds, tissue targeting compounds, and biosensors.
  • microspheres enhances their dispersion in aqueous media, thereby enabling safe administration of the microspheres to animals, humans, or other biological organisms such as insects and plants in much greater amounts than conventional hydrophobic microspheres which require the presence of potentially biologically dangerous amounts of surfactants to achieve similar concentrations of administrable microspheres.
  • Some of the unique properties of these microspheres of the present invention are described below.
  • the microspheres are readily prepared from a wide variety of protein-containing compositions, i.e., albumin, casein, enzymes, protein-polysaccharide mixtures, protein-DNA compositions, protein-phospholipid compositions, etc.
  • the microspheres of the present invention exhibit smooth, round and uniform morphology.
  • microspheres of the present invention permits control of crosslinking density.
  • Any cross-linking agent such as aldehydes, formaldehyde, glutaraldehyde, carbodiimide or others known to those skilled in the art may be used at concentrations of from about 0.1% to 5% by weight.
  • the microspheres of the present invention may be prepared with high loading concentrations of compounds, from 0% to as high as 60% by weight. This is achieved by performing synthesis at various temperatures including low temperature, room temperature and high temperature to enable use of labile pharmacological compounds if coupled during microsphere synthesis and also by post-loading with a wide range of compounds after initial microsphere synthesis.
  • microwaves are used during microsphere synthesis.
  • This method may be performed in the absence of cross-linking agents or in the presence of very low levels of cross-linking agents.
  • This novel approach to microsphere synthesis decreases potentially toxic side effects of solvents and other chemicals used in previous methods of microsphere synthesis.
  • No glutaraldehyde, formaldehyde carbodiimide or other cross linker is required.
  • This method is not limited by the use of cross-linkers and the method is easier to control than prior art methods.
  • this novel method is not limited to specific functional groups such as amino groups as in previous methods and can use any functional group. Therefore, this novel method is not limited by the chemical experimental reaction conditions required by prior art methods.
  • An additional benefit of this novel methodology is that it is extremely rapid and produces uniform microspheres.
  • microspheres of the present invention are amenable to a wide range of aqueous and non-aqueous chemistries for loading or surface binding of compounds.
  • Such compounds include, but are not limited to, receptor ligands, antigens, antibodies, immune recognition molecules, growth factors, glycoproteins, carbohydrates, peptides, peptide conjugates, peptide analogs, and cell adhesion molecules.
  • Microsphere particle size diameters can be made from the low nanometer (nm) to high micrometer ( ⁇ m) range, for example from 1 nm to 50 ⁇ m. Microspheres of different sizes may be used for a variety of applications. In one embodiment, nm microspheres of the present invention may be used for intravenous (iv) administration.
  • microspheres may be employed for other modes of delivery such as topical, parenteral (pt), intramuscular (im), subcutaneous (sc), intrathecal (it), intracerebroventricular (icv), intraperitoneal (ip), gastrointestinal, urinary, oral, anal, vaginal, and aerosol delivery.
  • pt parenteral
  • im intramuscular
  • sc subcutaneous
  • sc intrathecal
  • icv intracerebroventricular
  • ip intraperitoneal
  • gastrointestinal urinary, oral, anal, vaginal, and aerosol delivery.
  • microspheres of the present invention may also be delivered as a spray or in an aerosol.
  • delivery methods may be useful in topical application to the skin of animals and humans, for delivery to fields of crops as fertilizers, herbicides and other agricultural uses, and for pesticide application to insects.
  • microspheres of the present invention are readily lyophilized for prolonged stable storage with subsequent ease of aqueous dispersion for administration.
  • Microspheres may be designed and made using the present invention to dissolve at specific pH levels.
  • microspheres may be designed to dissolve at a selected pH and remain stable at another pH, thereby providing a range of stability and storage options. This feature of the present invention also facilitates design of targeted delivery of compounds through changing pH conditions. For example, a microsphere may be stable at neutral pH and then dissolve at the acidic pH conditions found in the stomach, thereby facilitating delivery of orally administered compounds.
  • the release of compounds from the microspheres of the present invention may controlled by designing compound-binding properties, such as covalent, and ionic properties, and also by designing cross-linking densities which affect porosity and rate of biodegradation.
  • microspheres of the present invention may be avidly taken up by phagocytic cells for targeting intracellular infections and for presentation to cells of the immune system.
  • Intracellular infections might reside within macrophages and be targeted with the microspheres of the present invention.
  • Intracellular infections include but are not limited to HIV and hepatitis.
  • Microspheres comprising albumin, casein, polyglutamic acid, carboxymethylcellulose, hyaluronic acid and numerous other proteins, polypeptides, polysaccharides, phospholipids, polynucleotide components, or combinations thereof, found within the microsphere structural matrix are readily prepared by this method to form spherical microspheres with nanometer (nm) or micrometer ( ⁇ m ) diameters.
  • the microspheres of the present invention may be designed to comprise one or more compounds. These compounds may be loaded during or after microsphere synthesis. In another embodiment of the present invention, microspheres may be designed for loading with one or more compounds both during and after microsphere synthesis. Accordingly, the microspheres of the present invention may contain one or more compounds within the microsphere and one or more compounds on the surface of the microsphere.
  • microsphere synthesis is achieved by a steric stabilization process which creates aqueous dispersions of the matrix macromolecules in an organic phase.
  • the organic phase is usually a hydrophobic polymer solution.
  • any hydrophobic polymer may be used in the practice of the present invention including, but not limited to, polystyrene, polymethylmethacrylates, cellulose acetate butyrate, and polyethylmefhacrylates.
  • the hydrophobic polymer solution is approximately a 2: 1 ratio of chloroform to toluene.
  • cellulose acetate butyrate is mixed in 1,2-dichloroethane at a concentration of about 4 g/200 ml.
  • a cross-linking agent for example, glutaraldehyde (25% stock solution) added to an organic solvent such as toluene or hexane (each at 100%), is introduced into the organic phase to first stabilize a lightly cross-linked particle structure.
  • a cross-linking agent for example, glutaraldehyde (25% stock solution) added to an organic solvent such as toluene or hexane (each at 100%)
  • an organic solvent such as toluene or hexane (each at 100%)
  • microspheres may then be cross-linked further to the extent desired, depending upon the amount of cross-linker used. While not wanting to be bound by this hypothesis, it is believed that the amount of agitation affects the microsphere particle size. It is to be understood that the amount of agitation and sonication may be varied depending on the specific microspheres to be synthesized and the compounds to be loaded or post-loaded.
  • a mixture of toluene and glutaraldehyde (25% stock solution) is used in a ratio of about
  • glutaraldehyde is mixed with 1,2 dichloroethane at about 40 g of glutaraldehyde (25% stock solution) in 30 ml of 1,2-dichloroethane.
  • glutaraldehyde and formaldehyde in dichloroethane at about 20 g of glutaraldehyde (25%) and about 20 g of formaldehyde in about 30 ml of 1 ,2- dichloroethane.
  • Yet another embodiment is approximately a 1 : 1 mixture of glutaraldehyde (25% stock solution) in H 2 0 followed by about 30 ml of 1,2 dichloroethane.
  • Another novel aspect of the present invention is the addition of cross-linkers during the sonication cycle.
  • microspheres prepared with the methods of this invention retain numerous reactive functional groups, including but not limited to, aldehyde, carboxyl, thiol, hydroxyl, amino, and imino groups which are readily capped with a variety of therapeutic, agricultural and cosmetic compounds.
  • the term "capped” is used herein to indicate occupancy or binding of the functional group to a compound.
  • This capping chemistry may also be used to widely alter the surface ionicity, charge properties, hydrophilicity, and biospecific affinity properties of the microsphere compositions.
  • microsphere synthesis Since the conditions of microsphere synthesis are very mild (aqueous, neutral, low temperature), relatively labile compounds such as biopolymers and drugs may be physically trapped or chemically bound into the microsphere structure during synthesis.
  • Preferred temperature conditions may range from about 4° C to 37° C, although other temperatures may be employed.
  • a wide range of pH conditions may be employed with the microwave method of preparing microspheres, depending on the materials used for microsphere synthesis and the compounds to be loaded or post- loaded. This novel feature of the present invention provides significant versatility in microsphere design and synthesis.
  • the somewhat porous hydrophilic microsphere structures also exhibit the ability to readily load compounds after synthesis.
  • Compounds may be loaded within the microspheres and also on or in their surface. This process of "post-loading", usually from aqueous solutions, enables achievement of high and stable compound loadings of about 60 wt %.
  • the specific reaction conditions of pH and temperature involved in post-loading microspheres with compounds depend to a large degree on the compound to be loaded. With certain compounds, for example, insulin, an alkaline pH condition is desirable whereas other compounds may require an acidic or neutral solution condition.
  • the specific reaction conditions involving the total duration of exposure to microwaves also vary depending on the compounds and the specific materials used to make the microspheres. For example, durations of 1 second to 5 min may be employed depending on the power and pulse duration of the microwave energy. In the examples of the present invention, microwave energies of about 700 watts are employed. It is to be understood that other wattages of microwave energy may be employed, and that the exposure to microwaves may be continuous or pulsatile as desired.
  • Different compounds may vary in their stabilities and labilities upon exposure to the reaction conditions. It is to be understood that the conditions (matrix materials, time, pH, intensity of mixing, temperature, amount of microwave energy and characteristics and duration of exposure) may all be varied accordingly to produce microspheres of desired size and containing the desired compound or multiple compounds.
  • loading of compounds such as nucleic acids into microspheres may require use of a positively charged protein or proteins as the matrix of the microsphere due to the negative charge on the nucleic acids. This may be accomplished by altering the charge characteristics of BSA or by adding a charge component to the matrix.
  • the microsphere matrix may require use of a phospholipid gel in addition to BSA.
  • microsphere matrix may require attachment of other functional groups which may be accomplished through chemical modification or by use of a different matrix component such as a polypeptide, several polypeptides, or a mixture of selected synthetic peptides.
  • Clinically practical microspheres for intracellular compound delivery are made possible by the combination of the following properties which can be achieved: sub-micron size; lyophilized stable powders; dispersions which are easily reconstituted; high drug pay loads; avid uptake by phagocytic cells; easy modification with antibodies, such as anti-CD34 antibodies or receptor ligands; and intracellular degradation to release active drugs or other compounds.
  • microsphere technology of the present invention Several applications have been tested with successful results. Among these, the use of the microsphere technology for chemotherapy drugs; anti-inflammatory drugs; antioxidants; antibiotics; various intracellular drugs; anticoagulation agents; enzymes and hormones, such as insulin.
  • the present targeted drug delivery system provides the capability to target the delivery of high concentrations of therapeutic agents to specific locations, such as cells within tumors, and to precisely control the rate of drug release.
  • the present invention increases chemotherapeutic efficacy and provides new options for treating a variety of diseases, including cancer, without incurring the toxic side effects resulting from systemic administration of conventional chemotherapeutic agents.
  • this approach may be modified to deliver a wide variety of other cosmetic, therapeutic and agricultural compounds to treat a variety of conditions in humans, animals, insects and plants.
  • the present invention provides novel microspheres which can be loaded with various compounds including cosmetic, therapeutic and agricultural compounds.
  • the present invention also includes a novel method involving the use of microwaves for making microspheres.
  • the present invention also includes methods for making microspheres which are loaded with compounds during synthesis of the microsphere.
  • the present invention also includes methods for making microspheres which are loaded with compounds after synthesis of the microsphere.
  • a feature of the present invention is that compounds to be loaded into or onto microspheres are not exposed to chemicals such as cross-linkers that might deleteriously affect the compound.
  • microspheres of the present invention can be designed to contain and release a specified amount of compound.
  • microspheres of the present invention can be designed to release a specified amount of compound at a specified rate and at a desired site.
  • the present invention also provides microspheres that incorporate compounds on or in their surface that bind to desired targets such as molecules, cells, tissues and organs for release of the compounds contained within the microsphere.
  • the present invention also provides microspheres that incorporate compounds on or in their surface that bind to desired targets such as cells, tissues and organs to affect the function of the target.
  • microspheres of the present invention may also be designed for binding to target cells and releasing compounds within the cells.
  • microspheres of the present invention may also be designed for binding to intracellular target sites within target cells and releasing compounds at these intracellular sites.
  • the present invention provides microspheres that contain therapeutic, and chemotherapeutic agents, for treatment of patients, including cancer patients.
  • the present invention provides microspheres that contain hormones for treatment of patients.
  • the present invention provides microspheres that contain insulin for treatment of patients with insulin insufficiency or diabetes.
  • a microsphere is to be loaded with a compound during microsphere synthesis
  • the compound is mixed into the BSA solution prior to mixing the BSA solution with the solution of 1,2-dichloroethane in cellulose acetate butyrate.
  • the mixture was vortexed for 5-10 minutes using a Vortex Genie 2 (Fisher).
  • about 0.25 ml of a 25% solution of glutaraldehyde (Fisher) in 1,2-dichloroethane (Fisher) 40 mg glutaraldehyde/ml 1,2-dichloroethane was added and the mixture was then vortexed for 15-30 minutes (min).
  • the pellet was resuspended in 5 ml acetone. Next, 30 ml of a solution of 1% BSA in deionized water was added. The mixture was incubated overnight while rotating at 70% power on the rotator (Glas-Col.) at room temperature. The next morning the microspheres were either centrifuged at about 7500 x g at room temperature, and dried by evaporation at room temperature or kept in solution of acetone or saline.
  • Alpha-amylase at 1200 units per mg of protein was loaded into microspheres at concentrations of 10 mg and 25 mg per 2 ml of BSA solution (BSA solution of 0.45 g in 1.5 ml water).
  • BSA solution BSA solution of 0.45 g in 1.5 ml water.
  • One unit of alpha- amylase produces 1 mg of maltose from starch in 3 min at pH
  • Alpha-amylase was added into the BSA solution (0.45 g BSA/ 1.5 ml deionized water) described in Example 1. Control microspheres were not loaded with alpha-amylase. Microspheres were air dried. Next, 0.05 g of microspheres were incubated at room temperature with moderate mixing (70% power) on a rotator in a total volume of 1 ml in a 1% starch solution (Sigma, St. Louis, MO). The conversion from starch to maltose was measured over time (0, 2, 4, 6, 24, 48, 78 hours 2 weeks, and 5 months) using Lugol solution (Sigma).
  • microspheres containing 25 mg amylase were active, and completely converted all the starch to maltose after 24 hours.
  • Microspheres loaded with 10 mg amylase converted all the starch to maltose after approximately 48 hours.
  • the microspheres containing 10 mg amylase and stored for 5 months in phosphate buffered saline at 4° C were inactive after 52 hours but the microspheres with 25 mg amylase showed complete conversion at 18 hours.
  • microspheres containing 10 mg or 25 mg amylase and stored for 5 months as a powder at 4° C were both active and each completely converted starch to maltose within approximately 24 hours.
  • microspheres used in this experiment were spun in a microfuge for 2 min at about 14,000 rpm, and stored in phosphate-buffered saline at 4° C for two weeks. These microspheres were then exposed to a 1% starch solution. Both microspheres containing 10 mg and 25 mg of alpha-amylase had observable activity after 24 hours to convert starch to maltose. Control microspheres without alpha-amylase displayed no observable activity. The results indicate good long term stability of enzymatic activity in microspheres made with this method.
  • the compound is mixed into the BSA solution prior to mixing the BSA solution with the solution of 1 ,2-dichloroethane in cellulose acetate butyrate.
  • the compound and BSA solution are mixed by vortexing on a high setting for about one minute. Any compound soluble in an aqueous environment may be used.
  • the mixture was vortexed for 5 to 10 minutes.
  • about 0.25 ml of a 25% stock solution of glutaraldehyde (Fisher) was added to 1 ,2-dichloroethane (Fisher) (40 mg glutaraldehyde per ml of 1,2-dichloroethane) and the mixture was vortexed for 15 to 30 min.
  • Also separately tested was an incubation of the mixture for about 1 hour at room temperature while rotating on a Glas-Col rotator at about 60% power.
  • a pellet was formed by adding approximately 40 ml of acetone and then spinning at about 7500 x g for 8 min. Acetone (Baychem Inc.) washes were done twice. After the final wash, the pellet was resuspended in 5 ml acetone. Next, 30 ml tetrahydrofuran (JT Baker) was added and incubated while stirring overnight at room temperature or heated (37° C) for 1 to 2 hours. Centrifugation was repeated at about 7500 x g for about 8 min followed by an acetone wash of the pellet. The pellet was resuspended in 5 ml acetone.
  • microspheres, without insulin were also made as a control. Approximately 0.005 g of microspheres loaded with insulin or not loaded with insulin (saline was the vehicle) were injected in a volume of approximately 1 ml, subcutaneously in the upper back, into Long Evans male rats (mean body weight about 297 g) and blood glucose readings were taken every hour for 6 hours and then at 24 hrs after injection. Three rats were injected with the insulin-containing microspheres (experimental group).
  • microspheres made with this method were injected intramuscularly into Long Evans male rats (mean body weight about 297 g) and blood glucose readings were taken.
  • the results of this study are presented in Figure 2.
  • the results demonstrate in vivo appropriate biological responses to insulin-containing microspheres as shown by the rapid reduction in blood glucose levels (Figs. 1 and 2).
  • microspheres of the present invention provide the capacity to employ much lower dosages of insulin without producing a long term hypoglycemia.
  • the mixture was then vortexed for 2 min followed by sonication at level 4 on ice for 1.5 min. Next add either 0.25 ml of a 50:50 mixture of 25% glutaraldehyde (Fisher)/40% formaldehyde (VWR) plus 1,2-dichloroethane (40 mg/ml) or a 50:50 mixture of 25% glutaraldehyde (Fisher)/water plus 1,2-dichloroethane (40 mg/ml) was added. Both of these formulations work but only one is added.
  • the mixture was sonicated (Ultrasonic Processor-XL, Environmental Safety Processes, Farmingdale, NY) for 30 seconds on ice at level 4 followed by mixing on a vortex for 15 min.
  • a pellet was formed by adding approximately 20 ml to 40 ml of acetone and then spinning at about 7500 x g for 8 min at room temperature. Acetone (Baychem Inc.) washes were done twice. After the final wash, the pellet was resuspended in 5 ml acetone. Next, 30 ml tetrahydrofuran (JT Baker) was added and incubated while stirring overnight on a Stirrer/hot plate (Fisher) at room temperature, or heated (37° C) for 1-2 hours.
  • JT Baker tetrahydrofuran
  • Centrifugation was repeated at about 7500 x g for about 8 min followed by an acetone wash of the pellet.
  • the pellet was resuspended in 5 ml acetone.
  • 30 ml of a solution of a 1 % BS A/water solution or a 1% proteolytically cleaved BSA/deionized water solution was added.
  • the mixture was incubated overnight at room temperature while rotating on a setting of high (80% power).
  • the next morning the microspheres were centrifuged as above and dried by evaporation or kept in solution at room temperature or at 4° C.
  • the microsphere technology has the unique aspect of loading large molecules, such as enzymes, hormones and other proteins, while maintaining their activities as shown in this study of loading acid phosphatase.
  • the levels of acid phosphatase in blood have important diagnostic implications for various disorders such as metastatic prostatic carcinoma, thrombocytopenia and liver disease.
  • Microsphere Synthesis and Assay Microspheres were synthesized by our previously described method.
  • Bulk loaded spheres (lot 9617) included 8.7% w/w enzyme/BSA while "post loaded” spheres (lot 9616) were capped with 5% w/w enzyme/BSA.
  • capped is meant attachment of the enzyme/BSA complex to free amino groups on the surface of the microsphere.
  • the presence and activity of the acid phosphatase was determined by use of Sigma Diagnostics® procedure number 104. Briefly, the assay involves incubating the samples in the presence of a substrate at 37° C for 30 min. The enzyme releases p- nitrophenol from the substrate in amounts proportional to activity. The addition of 0.1 M NaOH solution both quenches the reaction and develops the color. The absorbance of each sample is read in a spectrophotometer at 410 nm. This assay is insensitive to the presence of BSA or phosphate buffered saline (PBS).
  • PBS phosphate buffere
  • control enzyme solutions and microsphere suspensions were made. A sample of each solution and a sample of each microsphere suspension were removed and frozen. A 1 ml aliquot of each suspension was spun for 5 min at 3800 rpm, and an aliquot of the resulting supernatant was removed and frozen for subsequent analysis of enzyme activity. At 2 hours and at 9 hours later, a sample of the solution was removed and frozen. A 1 ml aliquot of each suspension was spun for 5 min at 3800 rpm and a sample of the supernatant was removed and frozen for subsequent analysis of enzyme activity. At 23 hours, a sample of each solution and each suspension was taken and heat treated in a water bath at about 60° C to 65° C for about 50 min.
  • the activity of the enzyme was assayed in either water or PBS.
  • the activity of the capping solution used to load the enzyme after sphere stabilization was tested both before and after treatment of microsphere lot 9616.
  • the release of enzyme from the microspheres was assayed by soaking the microspheres in water or in PBS for set time periods, taking and spinning down aliquots of the suspensions
  • samples of the enzyme solution and of each microsphere suspension were also heated in a water bath at 60° to 65° C for 60 min to determine if the microsphere provided protection to heat denaturation.
  • the pellet was suspended in 5 ml acetone. About 30 ml of tetrahydrofuran (JT Baker) was added and incubated while stirring overnight, or heated (37°C) for 1-2 hours. Centrifugation was repeated with acetone wash. The pellet was resuspended in 5 ml acetone. To 30 ml of either 0.1 % BSA/water/0.1 mg anti-CD34 monoclonal antibody (Immunotech, Coulter Co. FL) or 1 % BS A/water solution without antibody, 5 ml of microspheres in acetone was added and incubated overnight at room temperature rotating on high using a rotator (Glas-Col., Terre Haute, IN).
  • Rhodamine cube in a Nikon Diaphot-300 epi-fluorescence microscope Southern Micro Instruments, Atlanta, GA.
  • microspheres with and without antibodies were observed sticking to the outer surface of both cell types.
  • Microspheres containing CD34 antibody were observed inside KGIA cells due to the punctuate dots that appeared localized within the cells. The cells containing microspheres without antibody did not show this result. Cos cell staining was different then KGIA cells. Microspheres did not appear to be endocytosed and microspheres with antibody appeared to have better adherence than microspheres without antibody.
  • the resulting solution is called the microwave initiator and was stored in the refrigerator in an amber bottle.
  • bovine insulin Approximately 0.05 g of bovine insulin was mixed with 2 ml of the BSA solution plus 5 drops of 1 M NaOH and mixed. The NaOH facilitated entry of insulin into solution at pH 10. It is anticipated that another strong base will also favor entry of insulin into solution.
  • the insulin/BSA mixture was then added to the microwave initiator and stirred. Next, 10 ml of the DCE/CAB was added and the solution was vortexed. Then the solution was microwaved on high for 20 seconds. Controls contained all the same ingredients with the exception of insulin. The samples were then washed 3 times with 40 ml of acetone and air dried.
  • microspheres formed a whitish powder with particle sizes ranging from 10 ⁇ m to 30 ⁇ m for the microspheres without addition of NaOH, whereas microsphere size in the presence of NaOH was about 1 ⁇ m to 10 ⁇ m.
  • the final pellet was resuspended in 10 ml of saline.
  • a prepolymer solution is prepared by dissolving 12 g ammonium persulfate in 250 ml of 1 M HC1. The solution is then placed in a three necked flask and purged with nitrogen and cooled to 5° C. In a separate container, 21 ml of distilled purified aniline is mixed with 300 ml of 1 M HC1. The container is purged with pure nitrogen. The aniline solution is then added to the 3 necked flask. The mixture is cooled to 0° C and stirred for one hour to make the high molecular weight polyaniline.
  • the mixture is cooled to 0° C and stirred for 20 min to make the low molecular weight polyaniline.
  • the temperature of solution is then raised to 8° C to 10° C for 15 min.
  • the solution is cooled to 0° C and stirred for 45 min.
  • the polyaniline precipitate is then washed several times by filtration with distilled water. It is then treated with potassium hydroxide for 24 hours after which it is washed again for 6-12 hours in distilled water and dried in a vacuum oven for 24 hours at 50° C.
  • the mixture is optionally extracted with a soxalate extraction with acetonitrile for 3 hours until the extract is no longer colored. This extraction produces a polyaniline powder.
  • the polyaniline is dried in an oven at 50° C for 6 to 7 hours, followed by grinding to a powder.
  • Insulin Microspheres made without Protease-treated BSA Insulin microspheres were made according to the method of Example 8 although the BSA was not treated with protease. Microspheres of 30 ⁇ m to 50 ⁇ m in diameter were obtained.
  • Microspheres made with 6% and 10% Cellulose Acetate Butyrate
  • Example 8 The procedure of Example 8 was used except that the amount of cellulose acetate butyrate in 1 ,2-dichloroethane (CAB/DCE) was increased separately to 6% and 10%.
  • CAB/DCE cellulose acetate butyrate in 1 ,2-dichloroethane
  • Example 8 (0.45 g/1.5 ml deionized water) was added to 12 drops of the microwave initiator of Example 8 and then mixed by repeated refluxing for several minutes. Then 10 ml of 1,2- dichloroethane (Fisher)/6% cellulose acetate butyrate (Sigma) (DCE/CAB) was added and vortexed for 1 min to make an emulsion. The sample was sonicated for 1.5 min at a setting of
  • microspheres have also been prepared with this method although microwaves were applied for 20 seconds for
  • microspheres were sonicated on setting 4 for 1 min on ice. After the sonication step the microspheres were microwaved on a setting of high for 20 seconds. Next, approximately 40 ml of acetone was added and the microspheres were spun at 7500 x g for about 8 min. Acetone washes were repeated twice. After a final wash, the pellet was resuspended in 5 ml acetone resuspended by vortexing. Spherical microspheres of about 0.1 ⁇ m to 10 ⁇ m in diameter were produced.
  • microwave initiators are known as the following: 1) a short acting microwave initiator made with low molecular weight polyaniline; and 2) a long acting microwave initiator made with high molecular weight polyaniline.
  • the syntheses of these low and high molecular weight polyanilines were described in Example 8.
  • Three different microwave initiators were made by altering the ratio of short acting microwave initiator to long acting microwave initiator. These ratios are 1) 1 :3 ratio of short acting initiator: long acting initiator; 2) 1 : 1 ratio of short acting initiator: long acting initiator; and 3) 3: 1 ratio of short acting initiator: long acting initiator.
  • Solution A is a mixture of 3 ml polyaniline (using either high molecular weight polyaniline to form the long acting initiator or low molecular weight polyaniline to form the short acting initiator) in N-methyl pyrrolidone, combined with 20 ml of a 0.2% carboxymethylcellulose solution.
  • Solution B was made by mixing 10 ml of acetonitrile, 2 g p-toluenesulfonic acid and 1 ml H 2 0. Solutions A and B were combined in equal ratios.
  • 1,2-dichloroethane (40 mg/ml) was added followed by exposure to 5 second microwave bursts (at 700 watts) for a total of 20 seconds.
  • the microspheres were pelleted by addition of approximately 40 ml of acetone (Baychem Inc., Kennesaw, GA) and then spinning at about 7500 x g for 8 min.
  • Acetone washes were then done twice. After the final wash, the pellet was resuspended in 5 ml acetone.
  • the microspheres were centrifuged at about 7500 x g at room temperature and dried by evaporation at room temperature. A white powder formed. The resulting microspheres displayed variable sizes.
  • the pellet was resuspended in 5 ml acetone.
  • the microspheres were centrifuged at about 7500 x g at room temperature and dried by evaporation at room temperature. A white powder formed. The resulting microspheres displayed variable sizes.
  • microspheres made with these methods may be used for delivery of compounds, including but not limited to insulin.
  • Solution A A 20% casein solution was made by mixing casein (Sigma C5890) in 0.5M NaOH/H 2 0.
  • Solution B A 6% solution of cellulose acetate butyrate (CAB) was made in 1 ,2-dichloroethane (DCE), hereinafter called CAB/DCE
  • Solution C Glutaraldehyde saturated 1 ,2- dichloroethane was made in the following manner: about 10 ml of 25% glutaraldehyde was added to 30 ml of 1 ,2- dichloroethane, which was then vortexed for about 2 min until well mixed, allowed to settle until clear, centrifuged at about 1500 rpm for 5 min, and the supernatant was removed.
  • the mixture was sonicated on setting four for about 5 minutes.
  • the solution was aspirated through a 1.2 ⁇ m Nylon filter using a 60 cc syringe and transferred to a new container.
  • Solution C was added to a final concentration of 60 mg of solution C per ml of the previously aspirated solution and permitted to sit for 1-2 hours while rotating at 70% power. 5.
  • Acetone was added in an amount of about three times acetone per volume of this mixture, and allowed to sit overnight while rotating.
  • microspheres were ready for blocking agents or absorption of drugs, proteins or other substances.

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Abstract

Nouvelle composition et nouveau procédé permettant de fabriquer de nouvelles microsphères destinées à acheminer des composés jusqu'à des sites désirés in vivo et vitro. La présente invention concerne également des microsphères qui présentent des propriétés spéciales leur permettant de se charger de concentrations désirées de composés, de libérer des quantités désirées de composés à des vitesses désirées et de cibler la libération desdits composés sur des sites spécifiques. Elle concerne encore de nouvelles microsphères qui sont fabriquées à l'aide des micro-ondes et qui peuvent être utilisées pour l'administration de composés destinés à des applications cosmétiques, thérapeutiques et agricoles.
PCT/US1998/007560 1997-04-16 1998-04-16 Procede et composition pour l'administration ciblee de composes WO1998046210A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010010299A2 (fr) * 2008-07-23 2010-01-28 Yvon Gauthier Composition dermocosmetique, procede de traitement esthetique a partir de la composition, et utilisation de la composition pour eclaircir la pigmentation de la peau

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4431164A (en) * 1980-09-23 1984-02-14 Gesellschaft Zur Forderung Der Industrieorientierten Forschung An Den Schweizerischen Hochschulen Und Weiteren Institutionen Process and apparatus for producing microspheres
US5575987A (en) * 1992-09-02 1996-11-19 Takeda Chemical Industries, Ltd. Method of producing sustained-release microcapsules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4431164A (en) * 1980-09-23 1984-02-14 Gesellschaft Zur Forderung Der Industrieorientierten Forschung An Den Schweizerischen Hochschulen Und Weiteren Institutionen Process and apparatus for producing microspheres
US5575987A (en) * 1992-09-02 1996-11-19 Takeda Chemical Industries, Ltd. Method of producing sustained-release microcapsules

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010010299A2 (fr) * 2008-07-23 2010-01-28 Yvon Gauthier Composition dermocosmetique, procede de traitement esthetique a partir de la composition, et utilisation de la composition pour eclaircir la pigmentation de la peau
FR2934158A1 (fr) * 2008-07-23 2010-01-29 Yvon Gauthier Composition dermocosmetique, procede de traitement esthetique a partir de la composition, et utilisation de la composition pour eclaircir la pigmentation de la peau.
WO2010010299A3 (fr) * 2008-07-23 2011-04-28 Yvon Gauthier Composition dermocosmetique, procede de traitement esthetique a partir de la composition, et utilisation de la composition pour eclaircir la pigmentation de la peau

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